Int. J. Curr. Res. Biosci. Biol. 2015, 2(12): 75-79 International Journal of Current Research in Biosciences and Plant Biology ISSN: 2349-8080 Volume 2 Number 12 (December-2015) pp. 75-79 www.ijcrbp.com

Original Research Article

In Vitro Propagation of glutinosa (Lour) C.B. Robinson - An Endangered Medicinal Tree in Madhya Pradesh,

S.K.Tiwari1*, G. Krishnamurthy1, M.P. Goswami1, Amit Pandey1 and P.K. Singhal2

1Forest Genetics, Plant Propagation and Biotechnology Division, State Forest Research Institute, Jabalpur-482 028, Madhya Pradesh, India 2Rani Durgavati University, Jabalpur, Madhya Pradesh, India

*Corresponding author.

Abstract Keywords Litsea glutinosa (Lour) C.B. Robinson is a member of family and it is small tree commonly known as Maida lakdi. It is found through in India such as Andhra Pradesh, Madhya Pradesh, Chhitgarh, Orissa, Western ghat and outer Himalayas. In Madhya Pradesh this species has been reported in Hosangabad, Chhindawara, Anuppur, Mandla Balaghat Satna and Rewa districts in mixed forest areas, along with streams and hilly slopes. It was distributed throughout Madhya Growth regulators Pradesh but due to over exploitation it becomes endangered now. In vitro Litsea glutinosa propagation of the species has been achieved through nodal explants. The nodal explants of 1-2 cm length were inoculated on to MS medium with supplemented Propagation plant growth regulators, IAA and BAP for multiplication and IBA and NAA for rooting. Multiple shoots (3.5 shoots per explants with 5.07 cm length) were recorded after 50 days in the MS+IAA+BAP (2:3mg/lit.). Multiple roots (4.50 roots per explants with 4.50 cm length) were recorded after 70 days in the MS+IAA+IBA (0.1:2 mg/lit.). The plant survival rate was found 15% during hardening.

Introduction it becomes endangered now. It is found in mixed primary and secondary forest and thickets throughout Litsea glutinosa (Lour) C.B. Robinson is a member of India and in the outer Himalayas’ (Kirtikar and family Lauraceae and is well known for the evergreen Basu1981). As per the IUCN category the species is small tree commonly known as Maida lakdi, and found listed in endangered category (Mudgal et.al., 1997). at tropical and sub-tropical Asia (CSIR, 1998). Litsea glutinosa is found through in India such as Andhra Litsea glutinosa (Lour.) C. B. Rob., an evergreen or Pradesh, Madhya Pradesh, Chhitgarh, Orissa, Western deciduous tree that reaches 3-15 m. It is a polymorphic Ghats and outer Himalayas. In Madhya Pradesh this species. Its leaves are alternate and elliptical to oblong- species has been reported from mixed forest areas, along elliptical 3.5–10 × 1.5–11 cm, velvety (particularly when with streams and hilly slopes. It was distributed young) or glabrous. Umbels contain many small throughout Madhya Pradesh but due to over exploitation yellowish flowers, with 8-20 stamens for male flowers.

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Flowering occurs between March and June and fruits Planting material appear in September-October. Fruits are round and about 8 mm or less in diameter. The tree is able to reproduce The planting material cuttings were collected from vegetatively: over half of the stems are produced by different agro climatic zone of M.P. and planted in mist vegetative reproduction, mostly root-suckering (Rabena, chamber. Single nodal segment of 1.5-2.5 cm length 2010). were collected from cutting and used as explants. The explants were washed with 2% liquid detergent Extran Litsea glutinosa is a multipurpose, fast-growing tree. (MERCK) for 5-6 min solution to removing dust The leaves are chopped and soaked in water to make particles. This was followed by the treatment of 1% plaster in the Northern . While Litsea solution of Bavistin (BASF, Mumbai) broad spectrum glutinosa gives a poor timber due to a low wood density, fungicides for 5-10 min to minimize fungal it is often used as fuel (Rabena, 2008). The species is contamination and then, these were washed 3-4 time highly recalcitrant in nature as the seeds are very small distilled water. The washed explants were sterilized for in size and the seed germination is also very poor 1-9 min. with mercuric chlorite (Hi-Media, Mumbai, (Soerianegara, 1995). It is a good coppicer and new India). After the HgCl2 treatment explants were washed shoots sprouting from damaged adventitious buds with sterilized distilled water for 3-4 times under (called coppice shoots) are mainly used for propagation laminar air flow cabinet. Explants were inoculated in (Training manual, 2002). culture tube containing Murashige and Skoog (1962) with supplemented auxin and cytokinin. Culture was The conventional propagation is hampered due to low kept in culture room at 25±2o and light for 16 h photo seed viability and no rooting of vegetative cuttings period. Observation recorded every 10 days. (Rabena, 2010). Thus there is need for alternative in vitro propagation method for large scale multiplication, Results and discussion improvement and conservation of the species. The objective of the study was to develop a procedure for its The data presented in Table 1 indicated that HgCl2 micropropagation. concentration 0.1% and timing of the concentration is important factor for reducing contamination. The Materials and methods percentage of contamination–free explants varied from 30-100%. The lowest value (30%) was recorded with 4 The study was carried out in the plant tissue culture min. treatment (0.1%) whereas the highest value (100%) laboratory at State forest research, Jabalpur, India. was obtained with 7 min. treatment.

Table 1. Effect of HgCl2 (0.1%) treatment period on sterilization of nodal segments of Litsea glutinosa. Rate of contamination Treatment duration No. of Contamination free (After days of treatment) (min.) explants explants after ten days (%) 2nd 4th 6th 8th 10th 1 10 - 6 9 10 10 0 2 10 - 5 10 10 10 0 3 10 - 3 6 9 10 0 4 10 - 2 5 7 7 30 5 10 - - 1 4 5 50 6 10 - - - 1 1 90 7 10 - - - - - 100 8 10 - - - - - 100* 9 10 - - - - - 100** --Indicate no contamination; *Indicate culture death due to tissue killing; (* 60-80%)(**80-100%)

Shoot multiplication In vitro rooting

Shoot induction was recorded after 10 days of fresh culturing. Root induction was recorded after 20 days of sub culturing. But Multiple shoots (3.60 shoots per explants with 5.12 cm But Multiple roots (4.50 roots per explants with 4.50 cm length) were recorded after 50 days (Table 2) in the length) were recorded after 70 days (Table 3) in the MS+IAA+BAP (2:3mg/lit.). The shoot multiplication stage of MS+IAA+IBA (0.1:2 mg/lit.). The rooting stage of Litsea Litsea glutinosa is shown in Fig. 1(A). glutinosa is shown in Fig. 1(B).

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Table 2A. In vitro multiplication of Litsea glutinosa (Oneway ANOVA). Treatments/ 95% Confidence PGRs Std. Std. interval for Mean Parameter combination No. Mean Minimum Maximum Deviation Error Lower Upper IAA+BAP Bound Bound T1 (2:3 mg/lit) 3 3.5700 0.03000 0.01732 3.4955 3.6445 3.54 3.60 T2 (2:4mg/lit) 3 2.0000 0.10000 0.05774 1.7516 2.2484 1.90 2.10 Shoot no. T3 (2:5 mg/lit) 3 1.5200 0.02000 0.01155 1.4703 1.5697 1.50 1.54 Total 9 2.3633 0.93008 0.31003 1.6484 3.0783 1.50 3.60 T1 (2:3 mg/lit) 3 5.0700 0.07000 0.04041 4.8961 5.2439 4.99 5.12 T2 (2:4mg/lit) 3 5.2100 0.01000 0.00577 5.1852 5.2348 5.20 5.22 Length T3(2:5 mg/lit) 3 3.8700 0.01000 0.00577 3.8452 3.8948 3.86 3.88 Total 9 4.7167 0.63889 0.21296 4.2256 5.2078 3.86 5.22

Table 2B. In vitro multiplication of Litsea glutinosa (Post Hoc Tests, LSD). Dependent Mean Difference 95% Confidence Interval (I) Treatments (J) Treatments Std. Error Sig. Variable (I-J) Lower Bound Upper Bound T2 1.57000 (*) 0.05011 0.000 1.4474 1.6926 T1 T3 2.05000 (*) 0.05011 0.000 1.9274 2.1726 T1 -1.57000 (*) 0.05011 0.000 -1.6926 -1.4474 Shoot no. T2 T3 0.48000 (*) 0.05011 0.000 0.3574 0.6026 T1 -2.05000 (*) 0.05011 0.000 -2.1726 -1.9274 T3 T2 -0.48000 (*) 0.05011 0.000 -0.6026 -0.3574 T2 -0.14000 (*) 0.03367 0.006 -0.2224 -0.0576 T1 T3 1.20000 (*) 0.03367 0.000 1.1176 1.2824 T1 0.14000 (*) 0.03367 0.006 0.0576 0.2224 Length T2 T3 1.34000 (*) 0.03367 0.000 1.2576 1.4224 T1 -1.20000 (*) 0.03367 0.000 -1.2824 -1.1176 T3 T2 -1.34000 (*) 0.03367 0.000 -1.4224 -1.2576 * The mean difference is significant at the .05 level.

Table 3A. In vitro rooting response in Litsea glutinosa (Oneway ANOVA). 95% Confidence Treatments/ PGRs Std. Std. Interval for Mean Parameter No. Mean Minimum Maximum combination Deviation Error Lower Upper Bound Bound t1= IBA 1mg/lit. 3 2.0000 0.50000 0.28868 0.7579 3.2421 1.50 2.50 t2= IBA 2mg/lit. 3 4.0000 0.50000 0.28868 2.7579 5.2421 3.50 4.50 Root No. t3= IBA 3mg/lit. 3 3.0000 0.50000 0.28868 1.7579 4.2421 2.50 3.50 Total 9 3.0000 0.96825 0.32275 2.2557 3.7443 1.50 4.50 t1= IBA 1mg/lit. 3 2.0000 0.50000 0.28868 0.7579 3.2421 1.50 2.50 t2= IBA 2mg/lit. 3 4.0000 0.50000 0.28868 2.7579 5.2421 3.50 4.50 Root length t3= IBA 3mg/lit. 3 3.0000 0.50000 0.28868 1.7579 4.2421 2.50 3.50 Total 9 3.0000 0.96825 0.32275 2.2557 3.7443 1.50 4.50 t1= IBA 1mg/lit. 3 3.6500 0.05196 0.03000 3.5209 3.7791 3.62 3.71 t2= IBA 2mg/lit. 3 4.6133 1.62562 0.93855 0.5751 8.6516 3.64 6.49 Shoot length t3= IBA 3mg/lit. 3 6.2100 0.45211 0.26102 5.0869 7.3331 5.69 6.51 Total 9 4.8244 1.40225 0.46742 3.7466 5.9023 3.62 6.51

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Table 3B. In vitro rooting response in Litsea glutinosa (Post Hoc Tests LSD). 95% Confidence Interval Dependent Mean Difference (I) Treatments (J) Treatments Std. Error Sig. Lower Upper Variable (I-J) Bound Bound t2= IBA 2mg/lit. -2.00000(*) 0.40825 0.003 -2.9989 -1.0011 t1= IBA 1mg/lit. t3= IBA 3mg/lit. -1.00000(*) 0.40825 0.050 -1.9989 -0.0011 t1= IBA 1mg/lit. 2.00000(*) 0.40825 0.003 1.0011 2.9989 Root no. t2= IBA 2mg/lit. t3= IBA 3mg/lit. 1.00000(*) 0.40825 0.050 0.0011 1.9989 t1= IBA 1mg/lit. 1.00000(*) 0.40825 0.050 0.0011 1.9989 t3= IBA 3mg/lit. t2= IBA 2mg/lit. -1.00000(*) 0.40825 0.050 -1.9989 -0.0011 t2= IBA 2mg/lit. -2.00000(*) 0.40825 0.003 -2.9989 -1.0011 t1= IBA 1mg/lit. t3= IBA 3mg/lit. -1.00000(*) 0.40825 0.050 -1.9989 -0.0011 t1= IBA 1mg/lit. 2.00000(*) 0.40825 0.003 1.0011 2.9989 Root length t2= IBA 2mg/lit. t3= IBA 3mg/lit. 1.00000(*) 0.40825 0.050 0.0011 1.9989 t1= IBA 1mg/lit. 1.00000(*) 0.40825 0.050 0.0011 1.9989 t3= IBA 3mg/lit. t2= IBA 2mg/lit. -1.00000(*) 0.40825 0.050 -1.9989 -0.0011 t2= IBA 2mg/lit. -0.96333 0.79579 0.272 -2.9105 0.9839 t1= IBA 1mg/lit. t3= IBA 3mg/lit. -2.56000(*) 0.79579 0.018 -4.5072 -0.6128 t1= IBA 1mg/lit. 0.96333 0.79579 0.272 -0.9839 2.9105 Shoot length t2= IBA 2mg/lit. t3= IBA 3mg/lit. -1.59667 0.79579 0.092 -3.5439 0.3505 t1= IBA 1mg/lit. 2.56000(*) 0.79579 0.018 0.6128 4.5072 t3= IBA 3mg/lit. t2= IBA 2mg/lit. 1.59667 0.79579 0.092 -0.3505 3.5439 * The mean difference is significant at the .05 level.

Fig. 1: In vitro multiplication of Litsea glutinosa. surface of the roots. Plantlet placed on mixture sand, soil (A) Multiplication stage and FYM (1:1:1) were used for the hardening and covered with bottles for 2-3 days for maintaining humidity. The plant survival rate was found 15% during hardening. The temperature of the mist chamber was ranged between 35 ±5ºC with relative humidity of 80 to 90% (Fig. 1C).

Conclusion

(B) Rooting stage The present study, in vitro micropropagation protocol was developed through nodal explants of Litsea glutinosa. High frequency of multiplication of shoot after 50 days (3.5 shoots per explants with 5.07 cm length) was obtained from nodal shoot segment in MS medium with IAA+BAP (2:3mg/lit.).

(C) Hardening stage References

Abdullah CSIR, 1998. The Wealth of India. Vol. VI. New Delhi. Kirtikar, K., Basu, B., 1981. Indian medicinal and the nature of its genuine saponin. Phytochem. 2, 887-892. Mudgal, V., Khanna, K.K., Hajra, P.K., 1997. Flora of Hardening of in vitro regenerated shoots Madhya Pradesh. Vol.2. Botanical Survey of India, Calcutta. 681p. The in vitro regenerated shoots were carefully washed in Murashige, T., Skoog, F., 1962. Clonal crops through running tap water, so as to remove media from the tissue culture. Springer-Verlag, Berlin. pp.392-403.

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Rabena, A.R., 2010. Propagation techniques of Training Manual on ‘Propagation Technique of endangered sablot (Litsea gultinosa) Lour. C.B. Commercially Important Medicinal Plants’ Andhra Rob. Nat. Peer Rev. J. 5, 56-77. Pradesh State Forest Department, 2002. p. 34 and Rabena, A.R., 2004. Botanical description on Litsea p.98. glutinosa Lour. Rob. UPLB-CFNR, College, Laguna, Phillipines. Soeriangera, I., 1995. Litsea Lamk, In: Timber Tree, Minor Commercial Timber (Eds.: Lemmens, R.H.MJ., Soeriangera, I., Wong, W.C.). P. Rrosea Foundation, Boqor, Indonasie. pp.306-323.

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